Field of the Invention:
The present invention relates to a porcine epidemic diarrhea virus (PEDV) vaccine specific to the isolates currently endemic in the United States which are capable of reducing clinical signs of disease caused by PEDV. Due to the high mortality (up to 100%) in less than 10 day old piglets, the disease is of economic concern to the U.S. swine industry.
Description of the Related Art:
The porcine epidemic diarrhea virus is an enveloped, positive-sense single-stranded RNA virus that causes acute diarrhea, vomiting, and dehydration in pigs. It was first identified in Europe but has become increasingly problematic in many Asian countries, including Korea, China, Japan, the Philippines, and Thailand. In April of 2013, PEDV emerged in U.S. swine in the Midwest, swiftly spreading across the country. By October 2013, PEDV was detected in swine herds in 18 States. The economic impact of PEDV infection has already been substantial. North American isolates of PEDV have been identified (Huang, et al. 2013; Stevenson et al. 2013), however no fully licensed vaccine is commercially available in the United States. Accordingly, there is a continuing need to develop vaccines capable of protecting pigs against disease associated with PEDV. It would be advantageous to develop a vaccine that is effective against emerging North American PEDV strains which could be administered via a mucosal route (oral or intranasal) as well as via parenteral methods (e.g., intramuscularly, subcutaneously or intravenously).
PEDV is a member of the subfamily Coronavirinae of genus Alphacoronavirus (Bridgen et al. 1993) and was first identified in England in 1971 and later in other countries, such as Belgium, China, Hungary, Italy, Japan, Korea, and Thailand (Oldham J. 1972; Pensaert and De Bouck P. 1978; Chen et al. 2008; Nagy et al. 1996; Martelli et al. 2008; Takahashi et al. 1983; Chae et al. 2000; and Puranaveja et al. 2009). Other members of this family include Porcine Respiratory Coronavirus (PRCV), Hemagglutinating Encephalomyelitis Coronavirus (PHE), and Transmissible Gastroenteritis Virus (TGEV). Although PEDV and TGEV viruses are related and the clinical signs are very similar, there is no immune cross-protection.
PEDV is an enveloped virus possessing approximately a 28 kb, positive-sense, single stranded RNA genome, with a 5′ cap and a 3′ polyadenylated tail. (Pensaert and De Bouck P. 1978). The genome comprises a 5′ untranslated region (UTR), a 3′ UTR, and at least seven open reading frames (ORFs) that encode four structural proteins (spike (S), envelope (E), membrane (M), and nucleocapsid (N)) and three non-structural proteins (replicases 1a and 1b and ORF3); these are arranged on the genome in the order 5′-replicase (1a/1b)-S-ORF3-E-M-N-3′ (Oldham J. 1972; and Bridgen et al. 1993). The first three emergent North American PEDV genomic sequences characterized, Minnesota MN (GenBank: KF468752.1), Iowa IA1 (GenBank: KF468753.1), and Iowa IA2 (GenBank: KF468754.1), have the same size of 28,038 nucleotides (nt), excluding the polyadenosine tail and share the genome organization with the prototype PEDV CV777 strain (GenBank: AF353511.1). These three North American PEDV sequences shared 99.8 to 99.9% nucleotide identities. In particular, strains MN and IA2 had only 11 nucleotide differences across the entire genome.
The PEDV S protein is a type I glycoprotein composed of 1,383 amino acids (aa). The S protein can be divided into S1 (1-789 aa) and S2 (790-1,383 aa) domains based on its homology with S protein of other coronaviruses (Chang et al; 2002; Cruz et al, 1994; Godet, et al 1994; Jackwood et al. 2001; Sturman and Holmes; 1984; and Sun et al. 2008). The S protein in coronaviruses is a surface antigen, where it plays a role in regulating interactions with host cell receptor glycoproteins to mediate viral entry, and stimulating induction of neutralizing antibodies in the natural host. Thus the S glycoprotein is a primary target for the development of effective vaccines against PEDV.
The PEDV M protein is the most abundant envelope component playing an important role in the viral assembly process and also induces antibodies that neutralize the virus. Likewise the PEDV N protein, which binds to virion RNA providing a structural basis for the nucleocapsid, may also be important for induction of cell-mediated immunity (Saif, L. 1993).
The only accessory gene in PEDV is ORF3. While accessory genes are generally maintained in field strains, alteration of ORF3 is thought to influence virulence; cell culture adaptation has been used to alter the ORF3 gene in order to reduce virulence (Song et al. 2003). In fact, through investigation of the ORF3 gene, researchers have charted the emergence of new genogroups of PEDV in immunized swine herds in China since 2006. Phylogenic studies of these strains and the geographical reemergence of PEDV in China have demonstrated that those field strains causing devastating enteric disease differ genetically in ORF3 from the European strains and vaccine strains (Park et al. 2011).
It is well know that different strains of PEDV do exist with varying levels of virulence. During the 1980s and 1990s, PEDV was prevalent throughout Europe, in countries such as Belgium, England, Germany, France, the Netherlands, and Switzerland. The frequency of reported cases in Europe subsequently tapered off and/or the disease caused by PEDV was not of sufficient economic importance to start commercial development of a vaccine (Song and Park 2012). While outbreaks of PEDV have been documented in China since the 1980s, variant strains of PEDV emerging since 2010 associated with large-scale outbreaks of diarrhea have been more acute and severe. Thus the trial of vaccine development was mainly accomplished in Asian countries (Song and Park 2012). Variants emerging since 2010 have been reported as having 80-90% morbidity and 50-90% mortality in suckling piglets (Bi et al. 2012; Pan et al. 2012; and Li et al. 1012). Recent evidence suggests that the emerging virulent forms of PEDV in China may be a result of evolution of the live vaccine strains (Chen et al. 2010).
As an enteric disease affecting the pig's intestine, PEDV spreads via fecal-oral exposure. Contaminated trucks and equipment are frequent sources of infection to naïve animals. The clinical signs of PEDV infection are similar to transmissible gastroenteritis virus (TGEV) infection (Pijpers et al. 1993). In pigs three weeks of age and younger, clinical signs (including acute watery, diarrhea, vomiting, and dehydration) can be seen as soon as 24 hours after PEDV infection leading to 100% mortality. can appear. PEDV-infected feeder and grower pigs, as well as sows and boars, can develop diarrhea and vomiting. The animals can also show signs of anorexia and can be lethargic. The full impact on older pigs is yet to be determined, but reduced feed efficiency, additional days to market, and the susceptibility of infected animals to secondary infections is likely. For sows, reduced body condition may negatively impact reproductive performance. Reports have indicated that there are signs that PEDV could become endemic in North American herds, resulting in persistent diarrhea and other challenges.
The gross and histological changes in the gut of animals infected with PEDV are similar in the United States as those observed in China; essentially the virus destroys the villi of a pig's intestine so that there is a failure to absorb nutrients. Huang et al. 2012 reported that animals succumbing to the disease in the Minnesota and Iowa outbreaks had gross pathological lesions confined to the small intestine and that the small intestine was characterized by thin translucent walls distended with yellow fluid. Histological evaluations revealed regions of small intestines with villus blunting and fusion and minimal lymphoblastic infiltration of the villi of the lamia propria.
Huang et al. 2013 characterized three different strains of PEDV from outgoing outbreaks in the United States—one from Minnesota and two from Iowa, designated MN (GenBank accession No: KF468752) and IA1 (GenBank accession No: KF468753) and IA2 (GenBank accession No: KF48754), respectively. Huang's phylogenic survey grouped PEDV strains as falling into two distinct genogroups, designated genogroup 1 (G1) and genogroup 2 (G2). The significant changes in the N-terminal domain (NTD) of the spike gene differentiated genogroup 1 and 2. Huang et al. 2013 suggests that the second deletion region (DR2) in the N-terminal domain (NTD) appears to have a higher degree of antigenic change than DR1, suggesting that the emerging North American strains may be less “antigenically” related to the G1a vaccine strains.
Genogroup 1 includes at least three clusters 1a, 1b, and R. Subgroup 1a includes the early European, Chinese, and Korean isolates, e.g., prototype CV777 strain (Belgium, 1978, GenBank: AF353511.1) and strains LZC (Gansu, China, 2006; GenBank: EF185992) and SM98 (Korea, 1998; GenBank: GU937797.1). Subgroup 1b contains five strains—one from South Korea (the DR13 attenuated vaccine strain, GenBank: JQ023162.1) and the others from China linked by the common “genetic signature” 8-aa deletion in nsp3 and the large ORF3 deletion at the C terminus. Group “R” is associated with recombinants of the other genogroups. However, the newly emergent PEDV strains, including those arising in China since 2010 and in North America since 2013, belong to genogroup G2a. The Chinese strain AH2012 (GenBank accession no: KC210145) and the North American strains share several unique nucleotides changes and are clustered together in genogroup 2a. Nucleotide identity to AH2012 for strains MN and IA2 was 99.6% and for strain IA1 was 99.5%. Researchers have speculated that an AH2012-like virus was possibly transmitted to the eastern China regions and then transported to the United States and is most likely the closest ancestor to the North American strains. Members of the genogroup 2a share only approximately 96.9% similarity to the prototype PEDV strain CV777 of genogroup 1a (Bridgen, et al. 1993; Huang et al. 2013; GenBank: AF353511.1). As such, the attenuated PEDV vaccines based on the historical CV777-derived G1a strains or DR13-derived G1b strains may be antigenically less related to the newly emergent Chinese and North American G2a PEDV strains and therefore may be poor vaccine candidates.
A closely related North American isolate US/Colorado/2013 (GenBank Accession No: KF272920.1) has also been reported by Marthaler et al, 2013. Like the North American isolates above, the complete PEDV genome of CO/13 has a nucleotide identity of 96.5 to 99.5% with other complete PEDV genomes available in GenBank, with the highest nucleotide identity (99.5%) with Chinese strain AH2012 (GenBank Accession No. KC210145). It is a member of the 2a genogroup. Comparison of the complete genome of CO/13 to that of PEDV reference strain CV777, demonstrates that CO/13 contains a 1-nt insertion (at position 48) and deletions of 5 nucleotides in the 5′ UTR (at positions 73 and 83 to 86). This North American virus exhibits increased divergence within S1 at genomic positions 20,696 and 21,125 sharing only 82% nucleotide identity with several insertions/deletions.
Several PEDV vaccines, which differ in their genomic sequence, mode of delivery, and efficacy, have been developed. A cell culture adaptation of the European CV777 strain has been used in Asian countries where the PEDV outbreaks have been severe. These have been in use since the 1990s.
In the early 1980s Japanese researchers isolated a causative PED virus strain 83P-5 from the diarrhea of an infected pig. Kusanagi et al. 1989 isolated and adapted the strain in Vero cells. An attenuated virus vaccine of cell culture adapted PEDV (P-5V) (83P-5) has been used in Japan in sows since 1997. The 100th-passaged 83P-5 strain was licensed for use as an attenuated PEDV vaccine in Japan by Nisseiken Co., Ltd. (Sato et al. 2011). It has been reported that adaptation and attenuation of the 83P-5strain showed mutations in the extra-cellular portion of the S protein with sequence similarity to that of the attenuated DR13 strain (Sato et al. 2011; See Strain 83P-5 Spike gene sequence at 100th passage, GenBank: AB548621.1). Although this later Japanese vaccine is considered efficacious, not all sows were able to pass immunity to their piglets (Usami et al. 1998). The Japanese strains and the European strains are members of genogroup G1a or G1b. As discussed above these attenuated vaccine strains are less related to the divergent North American strains than the newly emergent Chinese strains of genogroup 2a.
Oral vaccination with an attenuated Korean PEDV strain, DR13 (passage level 100) (GenBank: JQ023162.1), a member of genogroup G1b, has been shown to be efficacious as a vaccine. The viral strain was licensed and used as an oral vaccine in South Korea since 2004, and registered and commercialized in the Philippines in 2011 (Song and Park 2012). However, it has been reported that attenuated DR13 does not significantly alter the duration of virus shedding in challenged piglets—an indication that immune protection is incomplete. Moreover, oral immunization with highly attenuated PEDV only conferred protection at very high doses of vaccine (Song and Park 2012).
Other known vaccines include SUISHOT® PT-100 (ChoongAng Vaccine Laboratories, South Korea) a combination killed PEDV and TGEV vaccine, and SUISHOT® PED a killed PEDV vaccine. The strain and subtypes offered through ChoonAng Vaccine Laboratories are unknown. Also Komipharm International Co., another South Korean company, offers a series of killed, live, and combination vaccines marketed under the tradename PRO-VAC® which include the PEDV strain SM98P of genogroup G1a. Qilu Animal Health Products Factory of China, also markets a combination killed vaccine in China containing PEDV and TGEV whose strain and subtypes are unknown.
Therefore, what is needed is a PEDV vaccine specific to the isolates currently endemic in North America which is capable of reducing the clinical signs of disease caused by PEDV, and inducing protective immunity in immunized animals, including the reduction of viral shedding in immunized animals.